The overall objective of the proposed research is to understand the molecular mechanisms of type I Interferon (IFN) gene regulation in response to virus infection. Human and mouse type IIFN genes are repressed prior to infection (pre-induction repression), induced, and switched off after induction (post- induction repression). The proposed grant aims at elucidating both specific biochemical mechanisms of IFN transcriptional control and a broader level of coordinating IFN signaling events. This will be accomplished using a variety of molecular genetic and biochemical approaches, including studies of mice in which the represser genes have been inactivated by homologous recombination and studies of cultured cells where the genes will be knocked down by siRNA. Human and mouse type I IFN genes are present in large clusters bearing a single IFN-beta and multiple IFN-alpha genes. Experiments are proposed to identify intergenic regulatory sequences and characterize cluster-wide changes in transcription factor binding, chromatin structure, and histone modification during virus infection. Cluster-wide changes in chromatin structure will be detected using chromatin precipitation (ChIP) and DNA microarray methods (chip). Regulatory sequences will be identified by patterns of chromatin modification, the use of nuclease sensitivity assays, and by studying intra-cluster interactions between putative regulatory sequences and promoters. The functional role of putative regulatory sequences will be determined using a molecular genetic approach involving "recombineering" of bacterial artificial chromosomes and insertion into mouse embryonic stem cells by homologous recombination. The antiviral signaling events that result from the transcriptional control of IFN are also mediated by distinct signal transduction pathways. The non-canonical I?B kinases TBK1 and IKK? play key roles in mediating this response. We propose to identify the distinct functions of these kinases through studies of mice lacking either or both of them. The proposed studies should provide significant advances in our understanding of the mechanisms involved in the coordinate and sequential activation of the type I IFN gene cluster and in the signaling pathways required to mount an anti-viral response. The results could lead to the development of treatments for infectious, inflammatory, and autoimmune diseases, while additionally identifying therapeutic targets for antiviral agents.